Table_3_Capsule Protects Acinetobacter baumannii From Inter-Bacterial Competition Mediated by CdiA Toxin.doc

Currently, Acinetobacter baumannii is considered as one of the most important infectious agents causing hospital acquired infections worldwide. It has been observed that many clinically important pathogens express contact-dependent growth inhibition (CDI) phenomenon, which modulates cell–cell and cell–environment interactions, potentially allowing bacteria to adapt to ever-changing conditions. Mainly, these systems are used for the inhibition of the growth of genetically different individuals within the same species. In this work, by performing cell competition assays with three genotypically different (as determined by pulse-field gel electrophoresis) clinical A. baumannii isolates II-c, II-a, and II-a1, we show that A. baumannii capsule is the main feature protecting from CDI-mediated inhibition. We also observed that for one clinical isolate, the two-component BfmRS system, contributed to the resistance against CDI-mediated inhibition. Moreover, we were able to demonstrate, that the effector protein CdiA is released into the growth media and exhibits its inhibitory activity without the requirement of a cell–cell contact. Lastly, by evaluating the remaining number of the cells pre-mixed with the CdiA and performing live/dead assay, we demonstrate that purified CdiA protein causes a rapid cell growth arrest. Our results indicate, that capsule efficiently protects A. baumannii from a CDI-mediated inhibition by a clinical A. baumannii V15 strain, which is able to secrete CdiA effector into the growth media and cause target cell growth arrest without a cell–cell contact.目前，鲍曼不动杆菌（Acinetobacter baumannii）被视为全球范围内引发医院获得性感染的关键致病菌之一。诸多临床重要致病菌均可表达接触依赖性生长抑制（contact-dependent growth inhibition，CDI）现象，该现象可调控细胞与细胞、细胞与环境间的相互作用，助力细菌适应不断变化的生存环境。此类系统的核心功能为抑制同物种内遗传背景相异个体的生长。本研究以3株经脉冲场凝胶电泳（pulse-field gel electrophoresis）分型的基因分型不同的临床鲍曼不动杆菌分离株（II-c、II-a及II-a1）为材料开展细胞竞争实验，证实鲍曼不动杆菌的荚膜是抵御CDI介导抑制的关键保护结构。此外我们发现，针对其中一株临床分离株，双组分调控系统BfmRS可增强其对CDI介导抑制的抗性。进一步研究表明，效应蛋白CdiA可释放至培养介质中，且无需细胞接触即可发挥抑制活性。最后，通过统计与CdiA预孵育的细胞残留数量并开展活/死检测实验，我们证实纯化的CdiA蛋白可快速诱导靶细胞生长停滞。本研究结果显示，荚膜可有效保护鲍曼不动杆菌免受临床鲍曼不动杆菌V15菌株的CDI介导抑制——该菌株可将CdiA效应蛋白分泌至培养介质中，且无需细胞接触即可引发靶细胞生长停滞。